The use of exosomes in clinical settings is progressively becoming a reality, as clinical trials testing exosomes for diagnostic and therapeutic applications are generating remarkable interest from the scientific community and investors. Exosomes are small extracellular vesicles secreted by all cell types playing intercellular communication roles in health and disease by transferring cellular cargoes such as functional proteins, metabolites and nucleic acids to recipient cells. An in-depth understanding of exosome biology is therefore essential to ensure clinical development of exosome based investigational therapeutic products. Here we summarise the most up-to-date knowkedge about the complex biological journey of exosomes from biogenesis and secretion, transport and uptake to their intracellular signalling. We delineate the major pathways and molecular players that influence each step of exosome physiology, highlighting the routes of interest, which will be of benefit to exosome manipulation and engineering. We highlight the main controversies in the field of exosome research: their adequate definition, characterisation and biogenesis at plasma membrane. We also delineate the most common identified pitfalls affecting exosome research and development. Unravelling exosome physiology is key to their ultimate progression towards clinical applications.

Exosomes are a subset of extracellular vesicles essential for cell–cell communication in health and disease with the ability to transport nucleic acids, functional proteins and other metabolites. Their clinical use as diagnostic biomarkers and therapeutic carriers has become a major field of research over recent years, generating rapidly expanding scientific interest and financial investment. Their reduced immunogenicity compared to liposomes or viral vectors and their ability to cross major physiological barriers like the blood–brain barrier make them an appealing and innovative option as biomarkers and therapeutic agents. Here, we review the latest clinical developments of exosome biotechnology for diagnostic and therapeutic purposes, including the most recent COVID‐19‐related exosome‐based clinical trials. We present current exosome engineering strategies for optimal clinical safety and efficacy, and assess the technology developed for good manufacturing practice compliant scaling up and storage approaches along with their limitations in pharmaceutical industry.

Over the last decade, pioneering liver‐directed gene therapy trials for haemophilia B have achieved sustained clinical improvement after a single systemic injection of adeno‐associated virus (AAV) derived vectors encoding the human factor IX cDNA. These trials demonstrate the potential of AAV technology to provide long‐lasting clinical benefit in the treatment of monogenic liver disorders. Indeed, with more than ten ongoing or planned clinical trials for haemophilia A and B and dozens of trials planned for other inherited genetic/metabolic liver diseases, clinical translation is expanding rapidly. Gene therapy is likely to become an option for routine care of a subset of severe inherited genetic/metabolic liver diseases in the relatively near term. In this review, we aim to summarise the milestones in the development of gene therapy, present the different vector tools and their clinical applications for liver‐directed gene therapy. AAV‐derived vectors are emerging as the leading candidates for clinical translation of gene delivery to the liver. Therefore, we focus on clinical applications of AAV vectors in providing the most recent update on clinical outcomes of completed and ongoing gene therapy trials and comment on the current challenges that the field is facing for large‐scale clinical translation. There is clearly an urgent need for more efficient therapies in many severe monogenic liver disorders, which will require careful risk‐benefit analysis for each indication, especially in paediatrics.